Nucléoside diphosphate kinase d'érythrocytes de bœuf: Caractéristiques cinétiques

Abstract

The catalysis of phosphate transfer from ATP to nucleoside diphosphates other than ADP is the physiological role currently devolved to the NDP kinases, especially to the one isolated from beef heart. Thus these enzymes allow the energy consumption needed for cellular syntheses to be supplied with the necessary UTP, CTP and GTP. We wanted to know if such is the physiological function of bovine erythrocyte NDP kinase which we purified. For that purpose we have undertaken the study of the reaction kinetics and of the inhibitions of this enzyme. Bovine erythrocytes and beef heart NDP kinases show similar kinetic properties : «ping-pong mechanism, substrates active as Mg-complexes, competitive inhibition occuring by excess of Mg-ADP substrate. Both enzymes also show similar kinetic and inhibition constants : KmMg⋅ATP=0,5mM,KmMg⋅ADP=0,15mMandKiMg⋅ADP=0,015mM Mg-ADP is a very strong inhibitor of this enzyme, so that it cannot act as a substrate in vivo. On the other hand, there is an important difference between both enzymes : Mg-ADP, though the strongest inhibitor, is not the only one acting on the erythrocytic NDP kinase. This could invalidate the usual interpretation of the physiological role of the NDP kinase with regard to the erythrocyte. In spite of the differences between beef heart and bovine erythrocytes NDP kinases, the molecular structure of these enzymes seems to be similar : their molecular weight is not very different (110 000), they have the same number of subunits (6 subunits having a molecular weights of about 17 000), and their phosphorylated form exhibits the same isoelectric point (pI = 6,0). La NDP kinase d'érythrocytes de bœuf présente des propriétés cinétiques et des constantes cinétiques et d'inhibition voisines de celles de la NDP kinase de cœur de bœuf : KmMg⋅ATP=0,5mM,KmMg⋅ADP=0,15mMandKiMg⋅ADP=0,015mM Par contre, des nucléosides diphosphates autres que l'ADP ont un effet inhibiteur sur la NDP kinase étudiée ; cette différence d'activité, reflétant des rôles physiologiques probablement distincts, semble aller de pair avec des structures moléculaires proches (poids moléculaire : 110 000 ; point isoélectrique : 6,0 ; nombre de sous-unités : 6).